ASTM E3285-22

This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the
Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
Designation: E3285 − 22
Standard Test Method for
Evaluation of Contact-Mediated Microbial Transference
This standard is issued under the fixed designation E3285; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope E1054PracticesforEvaluationofInactivatorsofAntimicro-
bial Agents
1.1 This test method is designed to evaluate the contact-
E2756Terminology Relating toAntimicrobial andAntiviral
mediated microbial transference on two-dimensional material
Agents
surfaces. Material surfaces intended to reduce microorganism
adherence or contamination may be evaluated using this test
3. Terminology
method.Additionally,thistestmethodcanbeutilizedonawide
3.1 Definitions—For definitions of terms used in this test
variety of material surfaces for other downstream processes.
method, refer to Terminology E2756.
1.2 This test method is designed to quantify the fomite
3.2 Definitions of Terms Specific to This Standard:
transferofbacteriaonmaterialsurfacesthroughtouch-transfer,
3.2.1 microbialtransference,n—relocationofamicroorgan-
be inexpensive, require limited training in practice, and be
ism from a previously contaminated surface to a different
adaptable to many surfaces or microorganisms. Non-porous
surface by contact.
surfaces are contaminated with inoculated filter paper as a
carrier and surface contamination is quantified by sampling
4. Significance and Use
with Replicate Organism Detection and Counting (RODAC)
4.1 This test method utilizes filter paper as a medium for
plates.
evaluating touch-transfer of bacteria to material surfaces.
1.3 Basic microbiology training is required to perform this
Sample surfaces are exposed to filter papers saturated with a
test method.
Staphylococcusaureussuspension,followedbyrecoveryofthe
bacteria from the surface with Replicate Organism Detection
1.4 The values stated in SI units are to be regarded as
and Counting (RODAC) plates. This test method reports the
standard. No other units of measurement are included in this
log reduction of bacterial transfer on an intended test surface
standard.
comparedtoacontrolsurface.Thetestandcontrolsurfacescan
1.5 This standard does not purport to address all of the
differ by texture, coating, treatment, or any other desired
safety concerns, if any, associated with its use. It is the
variables, as long as they are the same material.
responsibility of the user of this standard to establish appro-
priate safety, health, and environmental practices and deter- 5. Apparatus
mine the applicability of regulatory limitations prior to use.
5.1 Calibrated timer—readable in minutes and seconds,
1.6 This international standard was developed in accor-
5.2 Cell spreader—sterile,
dance with internationally recognized principles on standard-
ization established in the Decision on Principles for the 5.3 Centrifuge—capable of 2500 × g,
Development of International Standards, Guides and Recom-
5.4 Forceps—sterile,
mendations issued by the World Trade Organization Technical
5.5 Incubator—capable of maintaining a temperature of
Barriers to Trade (TBT) Committee.
35°C 62°C,
2. Referenced Documents
5.6 Thermometer or thermocouple,
2.1 ASTM Standards:
5.7 Variable volume pipettors and sterile tips, capable of
dispensing volumes from 10 µL-1000 µL, with current
This test method is under the jurisdiction of ASTM Committee E35 on
calibration,
Pesticides, Antimicrobials, and Alternative Control Agents and is the direct
5.8 Vortex mixer—any vortex mixer that will ensure proper
responsibility of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved March 15, 2022. Published April 2022. DOI: 10.1520/
agitation and mixing of the contents of test tubes.
E3285–22
For referenced ASTM standards, visit the ASTM website, www.astm.org, or 6. Reagents and Materials
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
6.1 Reagents:
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website. 6.1.1 Ethanol—commercially available 95% ethanol.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E3285 − 22
NOTE 2—Additional challenge species may be substituted to evaluate
6.1.2 Phosphate Buffered Saline (PBS)—Prepare according
the breadth of a material’s antimicrobial activity versus species to which
to manufacturer’s instructions to produce 1× PBS.
it may be exposed. If an alternative challenge species is used in testing, it
6.1.3 Trypticase Soy Agar with Lecithin and Polysorbate
must be identified in the final report, along with any accommodative
80—purchasedpre-madeplatesorpreparedaccordingtomanu-
modifications made to the method (that is, changes to culture media,
facturer’s instructions for a concentration of 45.1g⁄L and
buffers, etc.). The test report also must indicate that ASTM Test Method
E3285, modified, was used. The precision statistics reported in this
pipette 16mL into each sterile RODAC plate.
method will not apply.
NOTE 1—It might be necessary to perform a neutralizer effectiveness
test if there is a possibility of the material leaching antimicrobials. If
9. Preparation of Apparatus
deemed necessary, perform this test in compliance with Practices E1054.
9.1 Prepare flat test samples of the control and test surfaces
6.1.4 Tryptic Soy Agar (TSA)—purchased pre-made plates
by cutting into (50 6 2) mm × (50 6 2) mm squares. The test
or prepared according to manufacturer’s instructions for a
and control surfaces must be assayed at least in triplicate.
concentration of 40 g/L and dispensed into sterile 100mm ×
Sample size can be determined from preliminary data by
15mm Petri plates.
performing a power calculation.
6.1.5 Tryptic soy broth (TSB)—purchased pre-made or pre-
pared according to manufacturer’s instructions for a concen-
9.2 Adhere test and control surfaces to the inside bottom of
tration of 30g⁄L.
a 100mm Petri plate (with double-sided tape, if necessary),
ensuring that the test surface is facing up.
6.2 Materials:
6.2.1 Conical tubes—Sterile, 15mL and 50mL volume
9.3 Wipe surfaces with 95% ethanol 3 times, if compatible
capacity with threaded cap,
with surface.
6.2.2 Double-sided adhesive tape,
9.4 If surface is not compatible with ethanol, an alternative
6.2.3 Filter paper—sterilized cellulose, Example Whatman
methodofcleaninganddisinfectionmaybeused.Additionally,
grade 2, 8µm particle retention, 190µm nominal thickness,
2 samplecleaninganddisinfectionmaybeomitted,howeverany
97g⁄m nominal basis weight. Cut filter paper to two sizes,
modification to the cleaning and disinfection step shall be
45mm × 45 mm (+2 mm) and 60mm × 60 mm (+2 mm),
noted in the test report.
6.2.4 Inoculating loops—Sterile 10 µL,
6.2.5 Petri plates—sterile, 100mm diameter,
10. Procedure
6.2.6 RODAC plates—sterile, 65mm × 15mm,
6.2.7 Serological pipettes—Sterile, 10 mL and 25 mL
10.1 Inoculum Preparation:
volume,
10.1.1 Centrifuge culture from step 8.1.3 at 2500 × g for 5
6.2.8 Test tubes—Sterile, any with a volume capacity of
minutes at room temperature. Discard supernatant.
10mL, a minimum diameter of 16mm, and a cap that can
10.1.2 Resuspend cell pellet in PBS.
withstand 100 °C. Recommended size is 18mm × 150mm
10.1.3 Vortex thoroughly to generate a single-cell suspen-
borosilicate glass.
sion.
6.3 Test organism:
10.1.4 Dilute in PBS to reach a culturable cell titer of
3 3 4
6.3.1 Staphylococcus aureus, ATCC 6538
6.0×10 CFU/mL (range 1.0×10 to 1.2×10 ). Each sample
requires25mLofinoculum,sothisdilutionstepmaybescaled
7. Hazards
according to the number of samples tested. Cell density shall
be confirmed by serial dilution and plating onto TSA. This
7.1 Handling and manipulation of microorganisms that are
serves as a purity control, as colony morphology can be
potentially hazardous requires a high degree of technical
evaluated in the diluted samples.
competence and may be subject to legislation and regulations.
10.1.5 Check PBS for sterility by plating 100 µLof PBS on
Only personnel trained in microbiological techniques should
a TSA plate and incubate with the inoculation plates from
carry out such tests. Appropriate practices for disinfection,
10.2.3.
sterilization, and personal hygiene must be strictly observed.
NOTE 3—If necessary, the inoculum titer may be adjusted to produce
8. Culture Preparation
approximately100CFU/RODAC-250CFU/RODAConthecontrolspeci-
mensamples.Modificationoftheinocul
...